Airway constrictor hyperresponsiveness (AHR) - excessive airway narrowing in response to relatively minor stimuli - is characteristic of atopic and non-atopic asthmatic subjects, but also occurs in non-asthmatic individuals. Genetic linkage analyses suggest that overlapping but distinct gene sets regulate allergic inflammation or airway responsiveness. Together, these observations suggest that "native" (i.e., non-induced) AHR may reflect an underlying genetic predisposition to asthma that can occur independent of atopy, and that native AHR may be necessary, if not sufficient, for the development of clinical disease. The major objective of this proposal is to identify genes that control native AHR, and to understand how they do so, through genetic and physiological analyses of a recently established kindred of hyperresponsive BTBR strain mice harboring point mutations randomly induced by N-ethyl-N-nitrosourea (ENU). Over the last 3 years, we screened over 1300 generation 3 (G3) offspring of ENU-mutagenized BTBR mice, and identified a putative mutant that demonstrated reproducible, heritable airway cholinergic hyperresponsiveness, and whose abnormal phenotype has now been transmitted over 5 subsequent generations (to G8) in an autosomal dominant or semidominant inheritance pattern with incomplete penetrance. Importantly, our hyperresponsive mice do not exhibit the airway inflammation typical of asthma, and so their airway dysfunction represents an example of native constrictor hyperresponsiveness. We now seek to identify the genetic and physiological basis for abnormal airway responsiveness in this pedigree. Our rationale for studying this kindred of mutagenized mice, rather than existing isogenic strains, is that mutation(s) that caused their AHR is likely to have occurred in different genes from those at loci already identified as contributing to native AHR, e.g., in the A/J strain. This is so because the ENU-induced mutation(s) in this kindred can have occurred in any gene that regulates native airway constrictor responsiveness. Theoretically, exhaustive phenotypic evaluation of many thousands of mice harboring genome-wide, randomly induced mutations should many disclose different kindreds that together harbor mutations in virtually every gene which, in the context of the background strain, regulates native constrictor hyperresponsiveness. Our proposal represents the first step in such a systematic cataloguing of the genes that control airway responsiveness. Our straightforward experimental plan involves 2 interrelated Specific Aims in which we will: 1) identify the genetic alteration(s) responsible for abnormal AHR in our mutant mouse kindred; and 2) elucidate the structural and/or functional mechanisms through which the genetic alteration(s) lead to this abnormality. The proposed studies will reveal genes and mechanisms that control native murine airway responsiveness, and so should lead to better understanding of parallel mechanisms that might operate in human asthma and, perhaps, to development of improved therapeutic approaches intended to reduce AHR.